JP2016505269A - Method for selecting a single-cell organism in which a mutation has been induced, and a microfluidic device used therefor - Google Patents

Abstract

The present invention provides a microfluidic photoreaction apparatus and a method for sorting unicellular organisms having altered photoreaction characteristics. According to the present invention, the microfluidic system can be used to effectively use an improved single-cell organism based on phototaxis. It can also be easily monitored on a cell-by-cell basis, allowing mutant strains with altered photoreactivity and / or photosensitivity to be easily and via various analyzes including statistical analysis of collected results. It can be selected at high speed, and can be usefully used for elucidation of the correlation between phototaxis and light conversion efficiency, and improved single cell organism selection with improved light conversion efficiency.

Description

The present invention relates to a method for selecting a mutated unicellular organism and a microfluidic device used therefor, and more particularly, a method for selecting a mutated unicellular organism using phototaxis, which method includes a unicellular organism. If the chemotaxis index of the unicellular organism is changed compared to the step of irradiating the body with light to induce phototaxis, the step of calculating the phototactic index of the unicellular organism, and the control group, this Selecting as a unicellular organism in which the targeted mutation has been induced.

Various unicellular organisms including bacteria, yeast, and microalgae are used for various purposes in agriculture, livestock, fisheries, medicine and resources. For example, bacteria and yeast are widely used for the expression of pharmaceutical proteins. In particular, microalgae have the ability to produce a large amount of neutral lipids that can be converted into biodiesel from light energy, carbon dioxide, and inorganic substances. It is attracting attention as an alternative to solve the problem of energy resource depletion due to the rapid increase of fossil fuel consumption and global warming due to greenhouse gas emissions.

Microalgae contain pigments such as chlorophyll, carotenoids, and phycobilins, and unicellular algae that can synthesize cell growth and organic substances necessary for this through photosynthesis are called microalgae, and many plants Sex plankton belongs to this. To date, it has been reported that more than hundreds of thousands of microalgae are present in freshwater and marine ecosystems, and research and development have been attempted for various purposes. We are faced with many difficulties in improving strains.

For the efficient use of such microalgae, research for optimal strain development, medium optimization, optimal reactor design, metabolic process and product purification that meet the purpose is necessary.

One of these methods to develop the optimal strain is to induce specific or random mutations in the microalgae genome, followed by desired properties such as increased photosynthetic efficiency, high lipid production or rapid growth rate. It is to excavate a strain exhibiting characteristics.

US Patent Publication No. 2008-00254493 relates to a method for selecting a mutant microbial strain that does not express a proteolytic enzyme, and the cultivated strain is cultured on a gel containing a proteolytic enzyme temperament, It is disclosed to select mutant strains through the possibility of degradation.

Korean Patent Application Publication No. 2011-0018798 relates to a microfluidic cell chip, a cell death quantitative analysis method and a cell image analyzer using the same, and an apparatus capable of analyzing and imaging cell death in real time using a microfluidic system And a method are disclosed.

However, this often requires screening against tens of thousands of strains with complex biochemical and molecular biological analyses. Therefore, development of a method capable of selecting strains at a high speed in the initial stage of screening is required.
Therefore, as a result of diligent efforts to develop a method for efficiently selecting single-cell organisms by solving the above problems, the present inventors have used microfluidic photoreaction devices as a basis for phototaxis. It was confirmed that the improved single cell organism could be effectively selected, and the present invention was completed.

US Patent Application Publication No. 2008/00254493 Korean Patent No. 2011-0018798

An object of the present invention is to provide a method and apparatus capable of quickly and efficiently selecting an optimal single-cell organism in which a gene mutation having characteristics such as improved photoreactivity is induced.

In order to achieve the above object, the present invention includes (a) irradiating a single cell organism with light to induce phototaxis, (b) calculating a phototaxis index of the single cell organism, and (c) ) When the phototaxis index of the single cell organism is changed as compared with the control group, the mutant single cell using phototaxis comprising the step of selecting as a single cell organism in which the mutation intended for this is induced Provided are a method for selecting an organism and a mutant single cell organism selected by the method.

The present invention also includes a light transmissive individual inflow part, an individual reach part formed separately from the individual inflow part, a channel part connected to the individual inflow part and the individual reach part in fluid communication, There is provided a microfluidic photoreaction apparatus including a measurement unit formed between both ends of a channel unit.

FIG. 4 is a schematic diagram illustrating an overall process of selecting a photosynthetic strain having a specific photoreaction using phototaxis in a microfluidic system according to an embodiment of the present invention for a photosynthetic microalgae strain. 2 is a plan view of various exemplary microfluidic photoreactors used in the present invention. FIG. It is the top view (upper stage) and perspective view (lower stage) of the microfluidic photoreaction apparatus used in one implementation example of the present invention. 4 is a graph showing the photoreactions of microalgae measured using LEDs of various wavelengths according to an embodiment of the present invention in order to determine the wavelength of light effective for microalgae selection through phototaxis. FIG. 2A is a graph obtained by measuring the photoreaction of microalgae due to phototaxis using each microfluidic photoreaction device shown in FIG. 2A. It corresponds to the number indicated. It is the graph which showed ratio (a / b ratio) of the chlorophyll a and chlorophyll b in the microalgae strain | stump | stock used in one implementation example of this invention. FIG. 5 is a histogram showing the distribution of microalgae strains showing photoreaction with time using the microfluidic photoreaction apparatus according to an embodiment of the present invention in terms of the number of cells, and the wild type used as a control group. And the photoreaction degree of the mutant strain colony. It is the graph which analyzed the photoreactivity through the ratio of the cell number which showed the photoreaction with respect to the total cell number in each of a control group and a mutant strain. A graph showing the deviation of the average time required for the control group and the mutant strain to travel a certain distance (3 cm) by phototaxis, showing the photosensitivity between the mutant strains. The graph showing the correlation between the chlorophyll a / b ratio and NPQ shows that the higher the chlorophyll a / b ratio, the lower the NPQ (non-photochemical quenching) value, and the higher the photosynthesis efficiency. It is a graph showing the correlation between the chlorophyll a / b ratio and qP (Photochemical quinchin), and shows that the higher the chlorophyll a / b ratio, the higher the photosynthetic efficiency showing a higher qP value. In the correlation graph between the average arrival time and the photosynthesis efficiency measurement index NPQ in the phototaxis index according to one embodiment of the present invention, the mutant strain whose average arrival time is accelerated through the phototaxis is the photosynthesis efficiency measurement index A gold strain having a low correlation with a low NPQ value and increased photosensitivity indicates high photosynthesis efficiency. In the correlation graph between the average arrival time and the photosynthesis efficiency measurement index qP in the phototaxis index according to one embodiment of the present invention, a mutant strain whose average arrival time is accelerated through the phototaxis is a photosynthesis efficiency measurement index. A strain having a high correlation with a certain qP and having increased photosensitivity like NPQ indicates that the photosynthetic efficiency is high. A graph comparing the correlation between NPQ and the ratio of the number of moving cells showing the value obtained by dividing the number of cells that move a certain distance (3 cm) for a certain time (5 minutes) via the phototaxis by the number of cells in the control group It is. This is because a mutant strain having a value of 1 or more induced a mutation with excellent photosensitivity and photoreactivity by moving a larger number of cells compared to the control strain for a certain period of time. Such a strain shows a correlation with a low NPQ value, indicating that the strain has increased photosynthesis efficiency. It is an optical microscope (x40) image which shows the actual movement of the micro algae by a phototactic property using the microfluidic photoreaction apparatus which concerns on one implementation example of this invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used in this specification is well known in the art and widely used.

The present invention is based on the discovery that mutants having improved photoreactivity from photosynthetic unicellular organisms having motility can be effectively screened through differences in phototaxis, specifically, Specificity of light response to light through statistical analysis of individual movements of mutated cells in microfluidic systems using differences in light sensitivity and / or photoreactivity of single-cell organisms It was discovered that mutations with altered characteristics could be screened quickly and efficiently.

Accordingly, in one aspect, the present invention includes (a) a step of irradiating light to a single cell organism to induce phototaxis, (b) a step of calculating a phototaxis index of the single cell organism, and (c) A mutated single-cell organism utilizing phototaxis comprising the step of selecting a single-cell organism in which a mutation intended for this is induced when the photo-tactic index of the single-cell organism is changed as compared with a control group The present invention relates to a body sorting method.

The terms “single cell organism”, “cell” or “strain” as used in the present invention are used interchangeably, and are a variety of single cells that are motility, react to light, and exhibit phototaxis. It refers to organisms and includes, for example, protozoa or microalgae such as photosynthetic bacteria or bacteria, Euglena capable of photosynthesis. In one implementation, particularly the microalga, Chlamydomonas reinhardtii, is used.

The term "phototaxis" used in the present invention includes all positive phototaxis that moves along light by movement of a single-cell organism in response to light or negative phototaxis that moves by avoiding light. Even when the specific light quantity shows positive phototaxis, it may show negative phototaxis when the light intensity exceeds a certain intensity.

The term “mutation” used in the present invention means that a mutation occurs at the gene level compared to a control group in which no mutation was induced, and such mutation causes phenotype, particularly phototaxis, photoreaction. Differences are induced in characteristics such as gender and / or photosensitivity, and naturally occurring mutations include all artificially introduced mutations. Mutations include all mutations due to random or specific occurrences, additions, deletions, and / or substitutions of nucleotides constituting the gene.

The terms “target mutation” or “having target characteristics” and “mutation in which a target characteristic is induced” as used in the present invention shall be improved in a unicellular organism by genetic changes as described above. One or more characteristics are changed, eg, improved or improved, and include various characteristics depending on the end use of the unicellular organism. For example, when microalgae are used as unicellular organisms, characteristics or indicators related to photosynthesis, including changes in photosynthetic mechanisms including photosynthetic pigments, photosynthetic efficiency, photoconversion efficiency, other changes in growth rate, lipid content This includes, but is not limited to, changes in the amount and / or lipid component. To ascertain the degree of improvement, it can be compared with the corresponding properties in the mutated unicellular organism, and those skilled in the art can select appropriate criteria taking into account the improved characteristics. For example, compared with the control group, for example, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% From the above, it should be possible to select a single-cell organism in which a mutation aimed to be improved by about 80% or more, about 90% or more, or about 100% or more is induced.

Single-cell organisms used in the method of the invention for selecting individuals in which mutations exhibiting improved characteristics have been induced The natural or artificial mutation is induced and has one or more mutations in the genome It may be derived from a mutation library containing various unicellular organisms predicted to be The method of the present invention also includes a single colony derived from one cell, predicted to contain one type of mutation, or a number of cells derived from multiple cells each containing one or more of the same or different mutations. It may be derived from a colony. For example, if derived from a single colony, characterization, or selection of mutant single-cell organisms that exhibit optimal properties through individual characterization of single colonies, or mutation libraries when multiple colony mixtures are used Thus, a target mutation in which a gene mutation having a desired characteristic is induced can be used for efficient selection.

The method of the present invention is a rapid and simple analysis using phototaxis, and it is possible to detect several to tens of thousands of sudden mutations included in a mutant found in nature or a mutation library in which mutation has been artificially induced. During mutation, it can be effectively used to select individuals in which the desired mutation has been induced. In particular, in the latter case, the method of the present invention can be repeated to rapidly and efficiently select unicellular organisms in which a large amount of mutation has been induced.

The term “photoreactivity” used in the present invention refers to the property of moving to the opposite side of light by phototaxis when light is irradiated on a single-cell organism, and this is a fixed time, for example, 30 minutes. Measured with water of the total number of introduced cells, for example, about 3,000 cells that reached the opposite side by phototaxis when irradiated with a certain luminous intensity, for example, 30 μmol photon m ⁻² s ⁻¹ Can be done.

The term `` photosensitivity '' used in the present invention indicates how quickly the opposite side of the light moves due to phototaxis when light is irradiated on a single-cell organism, and has a constant light intensity, For example, when light of 30 μmol photon m ⁻² s ⁻¹ is irradiated, it can be measured by a time required to reach a certain distance, for example, 3 cm by the phototaxis.

The method according to the present invention is based on the reaction of a unicellular organism to light, and the phototactic reaction index or phototactic index used in the present invention is a characteristic change of a single cell organism in response to light. Indices that can be shown, can include a variety of numerical values, and can be calculated through one or more measurements of photoreactivity or photosensitivity. Such a phototactic index can include any ability to measure changes associated with response to light as compared to the control group, and (i) relative to the total number of single cell organisms used in the method of the invention. The ratio of the number of unicellular organisms migrated per unit time in response to phototaxis; (ii) a histogram vertex analysis based on the distribution of the number of unicellular organisms migrated per unit time; or (iii) used in the method Includes the average time, or speed, or deviation thereof required to move a single cell organism per unit distance. For example, it can be calculated by various methods described in the embodiments of the present invention and FIGS. For example, it is possible to observe the peak shift of the reaction arrival time of the wild type strain and the mutant strain through the vertex analysis of FIG. 5, and the ratio analysis of the cells that have reacted with the maximum reaction time of the cells through the vertex analysis, etc. Is possible.

In one implementation according to the present invention, when the present method is used in the apparatus according to the present invention, which will be described later, for example, (i) the total cell number used in the method for a certain period of time via the channel The ratio of the total number of cells that have migrated to (ii) the average time or deviation thereof required for a certain number of cells to migrate to the reach; (iii) the cells used in the method to the reach And (iv) including, but not limited to, the cell number distribution according to the time taken for the cells used in the method to move to the arrival part.

The unicellular organism that can be used in the method of the present invention is as described above. For example, a photosynthetic unicellular organism exhibiting phototaxis and motility, preferably microalgae, is used. In one implementation, the microalgae are green algae, diatoms, red algae, flagellates, pale green algae, brown flagellates, yellow green algae, dinoflagellates, or cyanobacteria, such as green algae (Chlorella, Dunaliella, Scenedesmus). , Haematococcus, Nannochloris, etc.), diatoms (Scheletonema, Thalassiosira, Phaeodactylum, Chaetoceros, etc.), red algae (Porphyris crusium, Galdieria, etc.) Algae (Chlamydomonas, Rhodomonas, Chromonas, etc.), yellow Color algae (such Olistodiscus), dinoflagellates (Crypthecodinium, Alexandrium, Gymnodinium, Chattonella, etc. Karenia), blue-green algae (Spirulina, Synechococcus, Synechocystis, etc. Cyanidium) but are exemplified, but the embodiment is not limited thereto. In one implementation, brown flagellar alga, preferably Chlamydomonas spp., Rhodomonas spp., Chromonas spp., More preferably Chlamydomonas reinhardtii, is used or not limited.

The method of the present invention may further include a pretreatment step of culturing the unicellular organism in a dark condition after culturing the unicellular organism in a continuous light condition before the step (a). The pretreatment step is to maintain the cell activity in the logarithmic phase state where the cell activity is the best by culturing under a continuous light condition. This is to increase sensitivity and increase photoreactivity of cells due to phototaxis.

That is, in the pretreatment step, the continuous light condition is not particularly limited as long as the amount of light that allows the microalgae growing through photosynthesis to be in the logarithmic phase state where the activity is most excellent is sufficient. Also, the light can be continuously irradiated for faster arrival in the logarithmic phase. As long as these conditions are satisfied, the amount of light and the irradiation time are not limited. For example, the continuous light condition is, for example, about 20 to 50 μmol photon m ⁻² s ⁻¹ intensity, preferably about 40 μmol photon m ⁻² s. ^{Although -1} intensity light is irradiated for about 12 to 24 hours, it is not limited to this.

Various ranges of light intensity and time may be used as long as the objective is achieved. The wavelength used may vary depending on the specific type of experimental single cell organism. Each single cell organism capable of photosynthesis has a wavelength of light that is efficiently recognized, and those skilled in the art should be able to select an appropriate wavelength in consideration of such matters. For example, in the case of the microalga Chlamydomonas, there is a part that senses light called eye spots, and the light wavelength that this part recognizes generally recognizes light having a wavelength of 540 to 600 nm or about 430 to 500 nm. Therefore, it is preferable to use light in this wavelength region band.

The culture in the pretreatment step is used in the next step after culturing until the exponential phase, which is the optimal state of the cell growth cycle. The growth of unicellular organisms is largely composed of an induction phase, an exponential phase, an log phase, a log phase, a growth phase; a stationary phase; and a death phase. One skilled in the art should be able to determine the logarithmic growth phase.

The phototaxis according to the method of the present invention includes both positive and negative phototaxis. Regarding the phototaxis, as described above, negative phototaxis is induced in the implementation example according to the present invention. At normal light intensities, in order to induce negative phototaxis in single cell organisms that induce positive phototaxis, intense light must be irradiated. That is, the intensity of light that can induce negative phototaxis varies depending on the target target organism, and light of various luminosities that can achieve such an effect is used. It should be possible to select a range of intensities. In a realization according to the invention, microalgae, in particular Chlamydomonas reinhardtii, are used, in which case the light intensity may be about 30 μmol photon m ⁻² s ⁻¹ cm ⁻¹ , Not limited. The wavelength of light is as described above.

Single cell organisms selected by the method of the present invention have increased photoreactivity and photosensitivity compared to a control group, i.e., a strain that has not been mutagenized, or a comparative strain used as a reference. Such a change in characteristics can be measured by the above-described phototaxis index, and can be selected as a strain in which a mutation aimed to improve the phototaxis index is induced. The degree of improvement of the phototaxis index varies depending on the analysis target or the type of the index, but for example, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, compared to the control group, Selecting as a single-cell organism in which a mutation is induced that is intended to be improved by about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more It should be possible.

The method of the present invention may include additional steps depending on the purpose of producing the mutant strain. For example, if mutation screening is for modification of photosynthetic characteristics, modification of lipid production, improvement of growth rate, it includes additional steps for each characteristic analysis. For example, a step of additionally analyzing a photosynthesis index, preferably a change in a photosynthesis mechanism including a photosynthesis dye, photosynthesis efficiency, and photoconversion efficiency may be included, but is not limited thereto. Such an analysis method is publicly known in the art, and a person skilled in the art can select an appropriate one. For example, various indicators described in the embodiments of the present invention and FIGS. 3 to 9, such as NPQ (non- including, but not limited to, photochemical quenching), or qP (photochemical quenching) and / or chlorophyll a / b ratio.

In another aspect, the present invention relates to a mutant single cell organism selected by the method for selecting a mutant single cell organism utilizing phototaxis.

The strain can be applied for production of useful substances in various fields according to the mode of mutation. For example, Chlamydomonas reinhardtii is one of the most studied species of microalgae to date, and genetic manipulations such as transformation are easier than other strains and related tools have been developed. At the same time, the genome sequence has been clarified, and it is considered to be a model organism of microalgae. Accordingly, mutant strains with improved photosynthesis mechanisms can be selected and used for lipid-related research, hydrogen production research, and the like for biodiesel production.

As described in the Examples and FIGS. 7 to 9, the strains selected by the method of the present invention have the target chlorophyll a / b ratio and the photosynthetic efficiency measurement indexes NPQ and qP through the analysis of the correlation. Mutation has been induced, indicating the excellence of the method.

In another aspect, the present invention is connected to a light transmissive individual inflow portion, an individual reaching portion formed separately from the individual inflow portion, and fluid communication with the individual inflow portion and the individual reaching portion. The present invention relates to a microfluidic photoreaction apparatus including a channel part and a measurement part formed between both ends of the channel part.

It will be apparent to those skilled in the art that the method of the invention can be used in a variety of devices as long as the method of the invention is accomplished. That is, as well as the apparatus of the present invention, other apparatuses capable of achieving such an object having portions corresponding to the respective configurations of the apparatus of the present invention may be used. Hereinafter, the configurations included in the apparatus of the present invention and the names thereof will be described by way of example, and should not be construed as being limited thereto, but should be construed as corresponding configurations in understanding and interpreting the invention. .

Referring to FIGS. 2A and 2B, the apparatus of the present invention includes an individual inflow portion 110 and an individual reach portion 120 formed at regular intervals, and various types of channels 130 are positioned therebetween. The individual inflow part 110 and the individual arrival part 120 are formed in a space, shape and size into which a single-cell organism to be analyzed can enter, and the target individual, for example, the size, characteristics and individual of the single-cell organism are used. Depending on the number, it can be formed in various shapes, sizes and / or materials. In order to see the reaction with respect to phototaxis, in one implementation it is made of a light transmissive material. The shape and size are not limited to a specific shape, but may be the same or different.

The channel is formed so that fluid can communicate with the individual inflow portion and the individual arrival portion. Through the channel, the culture solution and the single cell organism introduced together with the single cell organism move into the inflow portion of the individual. Therefore, the channel is fabricated with a structure and size that can minimize resistance so that the movement of a single cell organism is not disturbed. In one implementation, the channel is formed equal to or smaller than the diameter of the individual inflow portion or the reaching portion. In other implementations, it can have the same dimensions as described in FIG. 2B, but this is exemplary and not limiting.

A measurement unit 140 is formed at a part between both ends of the channel. Referring to FIG. 10, the measurement unit is a site for observing a moving single cell organism at a single cell level using a microscopic method, and has a structure and size capable of individually observing the movement of the single cell organism. Formed with. For example, on the basis of the diameter of the cell to be used in this apparatus, for example, it is manufactured in a size that allows passage of 1 to about 5 cells, and depending on the specific size of the target single cell organism used. change. For example, when about 10 μm to 200 μm, for example, the microalga Chlamydomonas is used, the diameter may be about 50 μm to 100 μm, but is not limited thereto.

Each configuration included in the microfluidic photoreaction apparatus of the present invention is not toxic to single cell organisms that are used with transparent materials that are light transmissive, is porous and easily transmits substances necessary for biological activity, and is a single cell organism. It is preferable to manufacture with the material which does not disturb the movement of a body, or the material pre-processed so that it may have the said characteristic. Examples of such a substance include, but are not limited to, PMMA (Polymethyl methacrylate), PS (Polystyrene), PDMS (Polydimethylsiloxane), and the like.

Referring to FIG. 1, the microfluidic photoreactor of the present invention may additionally include a light source. As long as the light source and wavelength capable of inducing the optimal phototaxis in the single-cell organism used in the apparatus of the present invention are selected and exhibit such effects, various light sources and wavelengths can be selected according to the object and purpose to be analyzed. Light is used.

Any type of light source can be used as long as it can emit a certain wavelength. For example, a laser diode or an LED (Light Emitting Diode) is used. In one implementation of the present invention, microalgae are used for analysis, and LED light sources that emit green and blue wavelengths are used, particularly in the case of Chlamydomonas reinhardtii.

FIG. 1 is a schematic view exemplarily showing an apparatus of the present invention and a mutant single cell organism using the same, that is, a strain selection method. The microfluidic photoreaction apparatus according to the present invention is used to select organisms whose characteristics of reacting to light are mutated by using the phototaxis of a unicellular organism having mobility that exhibits phototaxis. For example, a single cell organism derived from a single or multiple colony having a certain amount of phototaxis and motility, such as microalgae, is introduced into the cell inflow portion of the device according to the present invention, and then irradiated with light having a specific intensity Then, in response to this, the cell moves through the channel in a direction away from the direction in which the light source is irradiated. When the cells that move through the channel pass through the measurement unit, various data for the calculation of the phototaxis index, such as a certain distance travel time, the number of moving cells, etc. are collected through microscopic observation. In this regard, reference can be made to the above description. In the implementation example according to the present invention, when a microalgae is used as a unicellular organism in which a mutation utilizing phototaxis is induced, the following steps may be included.

For example, in one implementation, a method using the apparatus of the present invention includes, for example, (a) a pretreatment step of culturing a unicellular organism in a dark condition and then culturing the unicellular organism in a dark condition; ) Introducing the pretreated unicellular organism into the cell inflow portion of the photoreactor; (c) introducing into the cell inflow portion so as to induce phototaxis of the unicellular organism introduced into the cell inflow portion; Irradiating a light source; (d) observing the single-cell organism that moves to the arrival part through the channel by the phototaxis by the measurement unit and collecting a phototaxis reaction index; and (e) When the phototaxis index is changed as compared with the control group, the method includes, but is not limited to, selecting the cell from which the mutation has been induced. The pretreatment process may optionally be included. Reference may be made to the terms and descriptions used in the method referred to in connection with the inventive method described above.

Hereinafter, the present invention will be described in detail with reference to examples. These examples are merely for illustrating the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Production of a Mutation-Induced Strain The strain used in this example was obtained from Professor Chin-onson's laboratory at Hanyang University, and the species is Chlamydomonas renihardtii.
The strain is wild type strain JL428, which is a strain having a high chlorophyll a / b ratio, which is generally known after random mutation is induced in the wild type strain by insertion mutagenesis (insertion mutagenesis). Is highly likely to have high photosynthetic efficiency (Anastasis Melis (2012) Vol. 158 930-945), and a strain having a higher chlorophyll a / b ratio than that of the wild-type strain is selected first. Used for method efficacy verification.
The medium used for microalgae culture was TAP medium, and these constituents are shown in [Table 1].

Example 2: Microalgae culture and microfluidic device fabrication A microfluidic device was prepared by spin-coating the negative photosensitizer SU-8 50 on a silicon substrate, then covering the designed mask and exposing it to ultraviolet rays using an ultraviolet exposure machine. The polymer PDMS (Polydimethylsiloxane) and the curing agent were mixed at a ratio of 10: 1 and fabricated on the SU-8 mold fabricated by photolithography. . The completed PDMS microfluidic device was combined with a slide glass via plasma treatment. The manufactured device is as shown in FIG. 2B.

The device analyzed cell motility as a function of channel width and morphology as in Example 3 below to screen for optimal channel structure, as seen in FIG. 2A. In summary, while the green LED light source (540 nm) is illuminated on the part of the inlet cell inflow part containing the cells, the opposite part located at a certain distance, that is, the number of cells reaching the cell arrival part is measured with each device, As a result of comparing the results, as shown in FIG. 3B, a phenomenon was confirmed in which the movement due to the phototaxis of the cells was affected by the width of the channel adjacent to the cell inflow portion in the microfluidic device. Therefore, in order to facilitate the observation and statistical analysis of individual cells in the measurement unit while minimizing resistance to cell movement, the channel inlet width is changed from 4 mm to the outlet width of 100 μm as shown in FIG. 2B. A device design was selected that contained a constantly reduced form of channel and was fabricated as described above.

Example 3: Photoreaction analysis of microalgae according to the wavelength of light Artificial mutation in pretreatment process for adapting to efficiently and constantly adjust the photoreactivity and photosensitivity of cells to light The Chlamydomonas reinhardtii wild type strain (JL428) and the mutant strain of Example 1 were not seed-cultured in TAP agar medium. Specifically, the cells were cultured in a TAP liquid medium for 2 days under a light condition of 40 μmol photon m ⁻² s ⁻¹ intensity for 24 hours and a temperature condition of 23 ° C. After culturing for 2 days, when entering the logarithmic growth stage, it was diluted to 7.5 × 10 ³ cells ml ⁻¹ and stored for 1 hour in a dark room condition.

Thereafter, 40 ul of cells stored for 1 hour under dark room conditions are put into the cell inlet part of FIG. 2B below, and 40 ul TAP medium is put into the cell arrival part. The movement of algal cells was observed.

In order to analyze the photoreaction of microalgae by wavelength of light, the cell concentration and conditions were kept constant as described above, and then the wavelength of the LED light source used was adjusted to be different. Photoreaction analysis was carried out via the microalgae phototaxis under five conditions: green (540 nm), red (650 nm), blue (470 nm), white (full wavelength), and dark room conditions.

As a result, as can be seen in FIG. 3A, the microalgae showed a sensitive reaction at a specific wavelength, but showed a large reactivity in green (540 nm) and blue (470 nm), and in red (650 nm) to light. There was no reaction. From these results, a green LED light source having a wavelength of light of 540 nm basically used in the present invention was used.

Example 4: Photoreaction pattern analysis based on phototaxis of wild-type and mutant strains and selection Green LED light source (540 nm) is irradiated with light at 30 μmol photon m ⁻² s ^{−1 to} the cell inflow part, The number of cells reached was measured in minutes for 30 minutes. As a result, as shown in FIG. 5, as a result of analyzing the number of migrating individuals according to the time for each strain by photoreaction, a histogram of a certain form was obtained. As a result of comparing the histogram of the mutant strain with the histogram of the control group, it was confirmed that in the case of mutations 1, 2, and 3, the vertex of the photoreaction histogram moved to the left as compared with the control group. In contrast, mutations 4 and 5 did not show a significant difference from the control group. This result is related to the chlorophyll a / b ratio indirectly related to the photosynthetic efficiency. In general, a strain having a high chlorophyll a / b ratio is highly likely to be a strain having a high photosynthetic efficiency. Analysis of the histogram shows that the strain with an increased chlorophyll a / b ratio compared to the control group does not show a large difference between the control group and the chlorophyll a / b ratio due to an increase in the number of cells that migrate due to light. Since the mutant strains have almost no difference in the number of migrating cells due to phototaxis, it is easy and efficient to use the phototaxis reaction for a strain in which the mutation with increased photosynthesis efficiency is induced only by the pattern analysis of the histogram of FIG. It can be seen that this is possible.

In addition, as seen in FIG. 6A, as a result of comparing the photoreactivity of the control group strain and the mutant strain from the ratio of the number of individuals transferred to the total cell number versus the photoreaction, in the case of the control group strain, Compared to 65% showing a photoreaction, 85% of the mutation 1 migrated with a photoreaction, and about 80% of the mutations 2 and 3 showed a photoreaction. In contrast, mutations 4 and 5 showed similar ratios to the control group in the photoreaction ratio. This result indicates that mutant strains with increased photosynthetic efficiency due to increased chlorophyll a / b ratio have a greater degree of response to the same light intensity compared to the control group. In contrast, mutant strains that do not significantly increase the chlorophyll a / b ratio mean that the degree of photosynthetic efficiency is similar to that of the control group, and such strains also have photoreactivity to the same light intensity and control group. It means that it is similar.

The sensitivity to light can be analyzed comparatively by analyzing the deviation of the average time taken for cell migration within a certain time, based on the phototaxis of the control group and the mutant strain. It is possible to select specific strains. In the case of mutations 1, 2, and 3, it can be confirmed that the average time decreased as compared with the control group as seen in FIG. 6B. showed. This result means that mutant strains with increased photosynthetic efficiency due to increased chlorophyll a / b ratio are more sensitive than control strains to a certain light intensity, which is a measure of cell migration. Shown in relation to speed. Strains with increased sensitivity to light for the same light intensity show that the time required to move to a certain distance (3 cm) is reduced to a faster reaction. In the case of a strain having a relatively little difference in chlorophyll a / b ratio from the control group strain, the sensitivity to a certain light intensity is similar, and the time taken to travel a certain distance is similar to that of the control group strain. It shows that there is almost no difference.

On the other hand, as can be seen in FIGS. 5, 6A and 6B, the photoreactivity due to phototaxis of the microalgae control group and the mutant strain has a pattern very similar to the ratio of chlorophyll a / b shown in FIG. Shown, this shows that various indices (histogram apex, moving average time, ratio of photoreactive cells, etc.) obtained by the statistical analysis method for the individual photoreactivity of the strain applied in the present invention are the light due to phototaxis. It indicates that there is a possibility that it is related not only to reactivity and photosensitivity but also to changes in photosynthesis mechanism such as chlorophyll caused by mutation.

Therefore, it is possible to more efficiently search for a strain having improved photosynthesis efficiency for strains that have been primarily selected using the analysis method for the phototaxis of microalgae in the microfluidic system used in the present invention.

Example 5: Photosynthesis index analysis of selected mutant strains In this example, the photosynthetic efficiency of mutant strains selected by phototaxis as described above was measured. NPQ and qP were used as indices. NPQ is energy that cannot be used for photosynthesis in the photoenergy received for photosynthesis and disappears. The lower the NPQ value, the higher the photosynthesis efficiency, and qP means the energy used for photosynthesis. A higher value means higher photosynthesis efficiency.

5-1 Correlation analysis between chlorophyll a / b ratio of selected strains and NPQ, qP The correlation between chlorophyll a / b ratio of selected strains and NPQ, qP was analyzed as follows. Chlorophyll measurement was carried out by the published method based on absorbance measurement (Hartumut K. Richtenthaler and Claus Buschmann (2001) F4.3.1-F4.3.8).

In summary, chlorophyll measurement was carried out after entering the logarithmic growth phase after culturing at 40 μmol photon m ⁻² s ⁻¹ for 3 days in a flask. The flask was shaken well to transfer 1 ml to a 1.5 ml tube and centrifuged at 15,000 rpm for 1 minute. After centrifugation, the supernatant was removed and 1 ml of methanol was added, followed by vortexing to extract chlorophyll. Subsequently, the mixture was centrifuged at 15,000 rpm for 1 minute, and the absorbance was measured with respect to the supernatant at A6633.2 and A646.8 wavelengths. Then, the chlorophyll a / b ratio was measured by substituting into the following formula and measuring chlorophyll a and b.
Chla (g / ml) = 16.72 × A665.2-9.16 × A652.4
Chl b (g / ml) = 34.09 × A652.4-15.28 × A665.2
Chl a / b = Chl ÷ Chl b

NPQ and qP were measured using an Imaging-PAM chlorophyll fluorescence analysis equipment (Heinz Walz GmbH, Germany) after culturing the strain at 23 on an agar medium containing agar in a TAP medium.

Thereafter, the data obtained through the same analysis as in Examples 3 to 5, ie, the average migration time, the cell ratio of the cell population that reached the unit time, and the chlorophyll a / b ratio, NPQ and qP were analyzed. .

The results are described in FIGS. 7A and 7B. As shown, the higher the chlorophyll a / b ratio, the lower the correlation of the NPQ value and the higher the qP value, indicating that the photosynthesis efficiency is high.

5-2 Correlation analysis between average arrival time of selected strains and NPQ As described in Example 4, among the phototaxis indices obtained for each strain, the average arrival time and Example 5 Analysis with the photosynthetic efficiency measurement index described in 1.

The results are described in FIGS. 8A and 8B. As shown in this figure, the closer the r2 value indicating the correlation between the two indexes is, the higher the correlation is, and the higher the correlation is, the more the chlorophyll a / b ratio used in the prior art is, The index obtained in this way is useful for selecting mutant strains with increased photosynthetic efficiency.

5-3 Correlation analysis of the ratio of the number of migrating cells to the control group of the selected strain and NPQ As described in Example 4, among the phototaxis indices obtained for each strain, the control group Analysis of the ratio of the number of contrasting migratory cells and the photosynthesis efficiency measurement index described in Example 5 was performed.

The results are described in FIG. As shown in this figure, the mutant strain having a ratio of 1 or more migrated more than the control group strain for a certain period of time. By indicating an excellent mutant strain, this strain shows a correlation with a low NPQ value, indicating that this is a strain with increased photosynthetic efficiency. Therefore, such a result shows that a strain having high photosynthesis efficiency can be selected when the method of the present invention using phototaxis is used.

The present invention can effectively select single-cell organisms improved using phototaxis using a microfluidic system, and can be easily monitored on a cell-by-cell basis. Through various analyzes included, mutant strains with altered photoreactivity and / or photosensitivity can be selected easily or at high speed, elucidating the correlation between phototaxis and photoconversion efficiency, and improving photoconversion efficiency It can be used effectively for improved single cell organism selection.

Claims (24)

(A) irradiating light on a single-cell organism to induce phototaxis;
(B) calculating the phototaxis index of the unicellular organism;
(C) when the phototaxis index of the single-cell organism is changed as compared with the control group, the step of selecting as a single-cell organism in which the mutation intended for this is induced, A method for selecting mutant single-cell organisms. The method of claim 1, further comprising a pretreatment step of culturing the unicellular organism in a dark condition after culturing the unicellular organism in a dark condition before the step (a). A method for selecting a mutant single-cell organism as described. 3. The continuous light condition according to claim 2, wherein 20 to 50 μmol photon m ⁻² s ⁻¹ intensity light having a wavelength of 540 to 600 nm or 430 to 500 nm is irradiated for 12 to 24 hours. A method for selecting mutant single-cell organisms. The method for selecting a mutant single-cell organism according to claim 2, wherein the culture in the pretreatment step is performed until an exponential growth phase. The method for selecting a mutant single cell organism according to claim 1, wherein the single cell organism is derived from a single colony or a plurality of colonies. The method for selecting a mutant single cell organism according to claim 1, wherein the phototaxis is positive and negative phototaxis. The phototaxis is negative phototaxis, and light having a wavelength of 540 to 600 nm or 430 to 500 nm is induced by light having an intensity of 20 to 50 μmol photon m ⁻² s ⁻¹ cm ^−1. Item 7. A method for selecting a mutant single-cell organism according to Item 6. The method for selecting a mutant single cell organism according to claim 1, wherein the phototaxis index is calculated by solving at least one of photoreactivity or photosensitivity. The single-cell organism in which the target mutation has been induced is one of a growth rate of a photosynthesis index including a change in a photosynthetic mechanism including a photosynthetic pigment, a photosynthetic efficiency, or a photoconversion efficiency, as compared with a control group. The method for selecting a mutant single cell organism according to claim 1, wherein the above is improved. The phototaxis index is (i) the ratio of the number of unicellular organisms migrated per unit time in response to phototaxis to the total number of unicellular organisms used; (ii) the distribution of the number of unicellular organisms migrated per unit time A histogram vertex analysis based on: or (iii) an average time, or speed or deviation required for the movement of a single cell organism used in the method per unit distance, or a deviation thereof. A method for selecting mutant single-cell organisms. The method for selecting a mutant single-cell organism according to claim 1, further comprising a step of analyzing a photosynthesis index for the selected mutant single-cell organism. The method according to claim 11, wherein the phototaxis index includes a change in a photosynthetic mechanism including a photosynthetic pigment, a photosynthetic efficiency, or a photoconversion efficiency. The method for selecting a mutant single cell organism according to claim 1, wherein the single cell organism is a microalgae. The mutation according to claim 13, wherein the microalgae are green algae, diatom algae, red algae, flagellates, pale green algae, brown flagellates, yellow green algae, dinoflagellates, or cyanobacteria. Single cell organism sorting method. The method according to claim 14, wherein the brown flagellate algae is one or more of Chlamydomonas spp., Rhodomonas spp., And Chroomonas spp. 16. The method for selecting a mutant single cell organism according to claim 15, wherein the Chlamydomonas spp. Is Chlamydomonas reinhardtii. A sudden unicellular organism selected by the method according to claim 1. A light-transmitting individual inflow portion, an individual reaching portion formed separately from the individual inflow portion, a channel portion connected to the individual inflow portion and the individual reaching portion in fluid communication, and both ends of the channel portion And a microfluidic photoreaction apparatus including a measurement unit formed between the two. 19. The microfluidic photoreaction device according to claim 18, wherein the measurement unit is formed to be equal to or smaller than the diameter of the channel unit. 20. The microfluidic photoreaction device according to claim 19, wherein the diameter of the measurement unit is formed to a size that allows individual observation of the movement of the individual introduced into the individual inflow unit. The microfluidic photoreaction apparatus according to claim 19, wherein the diameter of the measurement unit is 10 to 100 m. 19. The microfluidic photoreaction device according to claim 18, further comprising a light source. 23. The microfluidic photoreaction device according to claim 22, wherein the light source is an LED (Light Emitting Diode) or a laser diode. The said apparatus is used for the method as described in any one of Claims 1-17, The microfluidic photoreaction apparatus of Claim 18 characterized by the above-mentioned.